Recombinant human proteins have become important pharmaceuticals. The combination of recombinant DNA technology and large-scale processes has enabled production of proteins that might otherwise have been impossible or too expensive to obtain from natural sources. For instance human growth hormone (hGH), immune interferon, tissue plasminogen activator (tPA) and human insulin are now commercial available by recombinant DNA technology.
However, the organism in large-scale bioprocesses is exposed to a changing environment and also respond to this. Differences in the production of recombinant proteins have been observed when increasing the reactor size. E.g. Riesenberg D et al. in Appl Microbiol Biotechnol 34:77-82, 1990, showed that the amount of human interferon alpha 1 was reduced by a factor of two when the culture volume was increased from 15 to 250 L.
When scaling up bioprocesses, differences in the micro-environment, such as concentration, and gradients, are likely to be expected.
Concentration gradients of substrates, such as the carbon source added at limiting feed (fed-batch), ammonia for pH titration and oxygen, are to be expected as the result of changing geometry and process parameters as well as the choice of feed position. Most organisms respond to rapid environmental changes and can also change their metabolism in a time-scale of seconds or less.
Pulse addition of glucose and the oscillating glucose concentration within the fermentor have earlier been studied with regard to yield and expression.
Pulse addition of glucose, i.e. addition of glucose within a few seconds when the culture had reached the steady state, was shown to induce gapA gene expression strongly and very rapidly (Gschaedler, Anne; et al, Biotechnol. Bioeng. (1999), 63(6), 712-720).
The effect of oscillating glucose concentration within the fermentor on biomass yield and acetate formation has been studied. The conclusion was, that the lower biomass yield and the higher acetate formation were caused by the cell response to the glucose oscillation within the fermentor when comparing large and small scale cultivations. (Bylund, F et al, Bioprocess Eng. (1999), 20(5), 377-389).
Pulse addition of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive pulses during fed-batch cultivation have positive effects on stabilizing plasmid and enhancing protein production. (Cheng, Chinyuan et al. Biotechnol. Bioeng. (1997), 56(1), 23-31). The pulse addition was repeated for 4-5 times at about 6 hours intervals. The result shows that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fed-batch cultivation, which would greatly reduce the product yield and limit the duration that the cultivation can be effectively operated.
Wang, Zhengjun et al. Biotechnol. Bioeng. (1993), 42(1), 95-102 have shown that a glucose pulse at the end of batch culture in YPD (rich complex medium) facilitated the transport of residual cytoplasmic invertase.
U.S. Pat. No. 5,912,133 discloses a fed-batch cultivation, wherein the carbon source concentration is at a constant low level under 5 g/L and wherein the carbon source is added in at least two feedings. The carbon source should be exhausted after the first feeding and before the second feeding.
It is also known to use external magnetic field with periodic variations and product stirring movement in a cell culture vessel. (DD271850). Thereby introduced material is effectively distributed with no local and potentially damaging concentrations
None of the reports disclose a repeated oscillation of organic carbon source in a square or sinus wave pattern, which can be characterized by amplitude and frequency. On the contrary it can be concluded from prior art that there should be a significant starvation period between two consecutive feedings (Cheng et al.) or that that a glucose pulse should only be done at the end of batch culture (Wang et al.).
The most applied technique to achieve high cell densities is the glucose-limited fed-batch. In a fed-batch process, all media components are supplied in excess as in a batch process, except for example the carbon source. A feed with a substrate solution, often glucose, is fed to the bioreactor with a rate that ensures that this substrate component is growth limiting. This substrate limitation allows control of the growth rate and the sugar uptake. By limiting the sugar uptake and thereby the reaction rate, engineering limitations, such as excessive heat evolution and oxygen limitation can be avoided. Glucose uptake can be divided in three reactions, glucose that is used for anabolism, glucose consumption for maintenance, which is the housekeeping requirement of the cell and glucose consumption to fuel growth. The yield in the last two reactions is approximately 1.07 gO2 gxe2x88x921sugar. Control of the growth rate can therefore be used to control the oxygen consumption and heat generation associated with growth. Furthermore, substrate limitation permits metabolic control by which overflow metabolism (i.e. acetate formation in case of E. coli) and catabolite repression can be avoided. In the baker""s yeast process sugar limitation is used throughout the process to avoid over-flow metabolism that results in excessive ethanol production (George et al. (1998). Bioprocess Eng 18: 135-142.). Inhibitory acetate production by E. coli is avoided in the same way (See e.g. Lee, S. Y. (1996). Trends Biotechnol 14: 98-105).
In the production of recombinant proteins the oxygen transfer and the dissolved oxygen tension is particularly important for the yield and quality of the product (See e.g. Bhattacharya and Dubey (1997) Enzyme Microb Technol 20: 355-360). A fed-batch process is therefore often the first choice when designing high-cell-density processes in E. coli.